DESCRIPTION: The goal of this proposal is to develop effective gene therapy strategies to treat hemophilia A (HemA) by direct in vivo bone marrow transfer of lentiviral vectors (LV) expressing human factor VIII (FVIII). Current treatment of HemA patients with repeated infusions of FVIII is costly, inconvenient, and incompletely effective. In addition, approximately 25% of treated patients develop anti-FVIII immune responses. Gene therapy treatment that can achieve long-term phenotypic correction without the complication of anti-FVIII antibody formation is highly desired. Stem cells (SCs) in the bone marrow are ideal targets for gene- and cell-based therapy because they are capable of self-renewal and can differentiate into blood and other types of cells. Intraosseous (IO) and neonatal delivery of lentiviral vectors (LVs) have been shown to effectively transduce bone marrow cells in mice. These approaches avoid the difficulties encountered by ex vivo SC gene transfer including maintenance of stem cell properties, the loss of engraftment potential after cell transfer, and negative effects of ex vivo cytokine stimulation. Furthermore, no manipulation of stem cells or pre-conditioning of the subject using potentially toxic, myelosuppressive agents is required using these approaches. Previously we have compared IO delivery of LVs utilizing two different promoters, a ubiquitous human elongation factor-1? (EF1?) promoter (E-F8-LV) and a human megakaryocytic-specific glycoprotein 1b? (GP1b?) promoter (G-F8-LV). We demonstrated that although SCs can be efficiently transduced by E-FVIII-LV, robust anti-FVIII immune responses were induced that eliminated functional FVIII in the circulation. In contrast, platelet-specific expression of FVIII as obtained following a single IO delivery of G-FVIII-LV, leading to long- term, partial correction of HemA in animals both with and without pre-existing inhibitors. Our findings suggest that platelets may comprise an ideal vehicle for delivering FVIII as FVIII stored in ?-granules is protected from neutralizing antibodies and, during bleeding, activated platelets locally excrete FVIII to promote clot formation. In the current proposal, we will test the hypothesis that: 1) IO delivery of a new G-DF8-LV construct incorporating a high-expressing FVIII variant, in combination with agents that suppress the initial innate immune response, will result in more efficient transduction of HSCs allowing both higher numbers of platelets expressing FVIII as well as higher levels of FVIII expression per platelet- thereby leading to effective treatment of HemA; 2) Phenotypic correction of HemA can also be achieved via IO delivery of a new N-F8-LV utilizing a mesenchymal SC (MSC)-specific nestin promoter (with or without immunomodulation). 3) By comparing neonatal delivery of E-F8-LV, G-F8-LV and N-F8-LV vectors, we will determine whether long-term phenotypic correction of HemA can be achieved by neonatal gene therapy. At the end of the study, we will be able to decipher the most effective LV system for directing BM SC gene therapy to treat HemA.